The effective application of gene therapy to heritable diseases of the hematopoietic system remains largely unrealized. Clinical studies demonstrate that the transplantation of allogeneic stem cells can correct many of these diseases. Therefore the goal of this project is to enhance gene transfer into marrow repopulating cells in baboons as a relevant, non-human primate, pre-clinical model for stem cell transplantation. Using a competitive repopulation strategy to study different variables related to gene transfer in baboons we have demonstrated 1) that Gibbon Ape Leukemia Virus-pseudotype vectors; 2) that efficient transduction of marrow repopulating cells can be accomplished by culturing cells from over the recombinant human fibronectin fragment CH-296; 3) that retrovirus mediated gene transfer is enhanced in G-CSF plus SCF primed (mobilized) peripheral blood and marrow progenitors. However, in spite of transient levels of marking in blood of between 10% and 20% during the initial post-transplant period, long-term gene transfer levels still are not high enough for most genetic diseases. Therefore, further improvements are needed in the transduction and then the engraftment of long-term repopulating stem cells. We propose to establish optimal methods for mobilization of repopulating cells that can be efficiently transduced and determine whether conditioning can be reduced without inhibiting in vivo repopulation by transduced cells. In addition, as the non-human primate model is logistically limited, we will determine whether identical information is provided using an immune-deficient mouse model. Finally, this project will extend findings from other projects. Studies will focus on the potential of novel hybrid vectors to transduce marrow repopulating cells and for ligands for the RAR and Notch families of receptors to enhance gene transfer into these repopulating cells. Non-human primates provide a highly relevant model in which these strategies can be directly tested before being introduced into human clinical trials.